2.7.8. Real‐time polymerase chain reaction (RT‐PCR) assay
RT‐PCR was used to study the effect of prepared scaffolds and PEMF on
the osteogenic gene expression ,including collagen type 1 (Col1),
Runt-related transcription factor 2 (Runx2), and Osteocalcin (OC). At
first, total mRNA was extracted using Qiazol reagent after 14 days of
culturing MSCs. Afterward, cDNA was synthesized by MLV reverse
transcriptase (RT) ,and random hexamer according to the manufacturer’s
instructions (Fermentas). The rate of gene expression was quantified by
an RT-PCR analyzer (Corbett, Australia) and Rotor-Gene software was used
to quantify the gene expression level. In this study, the expression of
the target gene was evaluated via the relative quantification model
compared to β2M control as a housekeeping gene. Relative gene expression
was calculated by ∆∆Ct method. The primer sequences of corresponding
genes are presented in Table 1.
Results and Discussion
In this study, we synthesized iron oxide nanoparticles showing
superparamagnetic properties. These nanoparticles highly tend to
aggregate ,which increases the overall size of the nanoparticles and
consequently precipitate them. Thus, surface modification seems
necessary to stabilize SPIONs and improve their biocompatibility
(Gupta & Gupta, 2005;
Hamley, 2003;
Ito et al., 2005). PAMAM dendrimer was
chosen as an appropriate surface modification because of its high water
solubility, biocompatibility, antimicrobial properties, and positively
charged amine groups (Chawla, Shetty,
Goyal, Rathore, & Sharma, 2018; Fox,
Richardson, & Briscoe, 2018).
The size and morphology of G3-SPIONs have been analyzed using the TEM
instrument and shown in Fig. 2a. The prepared G3-SPIONs had spherical
shape with the uniform size distribution. The average diameter of
nanoparticles was measured by the ImageJ software (NIH, Bethesda, MD)
and estimated to be 17.95 ± 3.57 nm. The three-dimensional structure of
dendrimers endues SPIONs steric stabilization, and thus well-dispersity
(Yen, Lien, Chung, & Yeh, 2017).
G3-SPIONs were also characterized by DLS ,which measures the
hydrodynamic diameter of the MNPs in their dispersion state (Fig. 2b).
The result showed that the average size of the nanoparticles was 28.91
nm ,which as expected, was more significant than the size obtained by
transmission electron microscopy (Souza,
Ciminelli, & Mohallem, 2016). The surface charge of G3-SPIONs ,which
is a crucial parameter for the electrostatic interaction between cells
and the surface of the material was assessed by zeta potential
measurement. The zeta potential of G3-SPIONs was 22.7 mV due to amine
groups in PAMAM dendrimer with a positive charge (Fig. 2c). Since some
of G3-SPIONs may be exposed on the nanofiber surface, the positive
charge can increase the cell attachment on the scaffold’s surface and
consequently improve cell-substrate interactions.
SPIONs functionalization and coating by APTES and PAMAM dendrimers,
respectively, on the surface of SPIONs were approved by FT-IR
spectroscopy, as shown in Fig. 3a. The characteristic absorption band at
586 cm-1 is attributed to the stretching bond of the
Fe–O which confirms the presence of
Fe3O4 nanoparticles. The peak at 1021
cm-1 is assigned to the stretching vibration of the
Si-O-Fe bond, proving the presence of SiO2 shell on the
surface of SPIONs. The peaks around 2866 cm-1 and 2912
cm-1 are due to the existence of stretching vibration
of CH2 in the aminopropyl group which confirms the
binding of APTES molecules. The broad peak at 3430
cm-1 is due to the stretching mode of the
NH2 group and the band at 1634 cm-1exhibits a stretching vibration of –CO–NH– group, representing the
binding of PAMAM dendrimers on the surface of SPIONs. It can be observed
that increasing the order of PAMAM generation increases the intensity of
these peaks.
The magnetization property of SPIONs ,and G3-SPIONs was measured using
VSM at room temperature . As shown in Fig. 3b, the negligible remanence
(Mr) and zero coercivity (HC) of the
magnetic nanoparticle confirm the superparamagnetic behavior of the
samples ,which prevents the formation of a stable magnetic state and
enables them to re-disperse rapidly in ,the absence of magnetic field.
compared with the maximum magnetization (MS) value of
68.28 emu/g for SPIONs, the Ms value for G3-SPIONs
decreases to 57.75 emu/g due to the linkage of non-magnetic PAMAM
dendrimer on ,the surface of SPIONs. Non- magnetic layer of PAMAM
inevitably lead to an unavoidable decrease in saturation magnetization.
It can result from a decrease in the strength of the exchange
interaction between oxygen and iron atoms
(Uzun et al., 2010). According to Taghavi
et al., the VSM result of bare MNPs analyzed at 37 ⸰C was obtained as
48.8 emu/g (POURIANAZAR, 2016) Zhu et al.
have prepared Poly peptide dendrimers through the ligand-exchange method
synthesized on MNPs. The saturation magnetization for G3(Lysine) and
G3(Glutamine) were reported 42.9 and 41.6, respectively. According to
recent studies, the saturation magnetization is related to the particle
size, directly. The reason for the decrease in saturation magnetization
arises from the increase in non-magnetic surface coverage of each
particle G3(Lys) and G3(Glu) as ligand-exchange
(Zhu et al., 2011). Considering the
mentioned studies, it could be concluded that the saturation
magnetization value of the SPION and G3-SPION is comparable with that
obtained in the previous studies.
The G3-SPIONs was embedded into the PCL nanofibers to be used as a
scaffold in the osteogenesis differentiation. The SEM micrographs of PCL
and G3-SPION-PCL nanofibrous scaffolds are presented in Fig. 4, to
analyze the diameter and morphology of the prepared scaffold. As shown,
the nanofibrous scaffolds have a cylindrical shape without any
structural imperfection resulting from the proper selection of
electrospinning parameters. The average fiber diameter of the PCL and
G3-SPION-PCL nanofibers obtained 866±310 and 495±144, respectively. It
was observed that the presence of G3-SPIONs in the scaffold has reduced
the size and distribution of fiber diameter ,which is justifiable by
higher conductivity of the polymer solution in the presence of
G3-SPIONs. High porosity and nanostructure property of the scaffolds
endue a high potential for cell attachment ,which can be used in tissue
engineering. Although the suitable average diameter of electrospun PCL
nanofibers for bone tissue engineering was reported in the wide range,
from 20 to 5000 nm, the average fiber diameter of the prepared
nanocomposite fibers was in the range of bone tissue engineering
applications, as confirmed by other studies
(Jang, Castano, & Kim, 2009;
Ren, Wang, Sun, Yue, & Zhang, 2017).
The MTT assay was used to investigate the proliferation rate and
viability of ADMSCs on electrospun PCL and G3-SPION-PCL scaffolds on
days 1, 3, 7, and 14. As shown in Fig. 5a, cell growth, and
proliferation have an increasing trend for all groups, indicating the
biosafety of the prepared scaffolds. As can be observed, there was no
statistically significant difference between the OD value of the cells
cultured on the PCL and G3-SPION-PCL on day 1. However, the difference
has been increased until the significant discrepancy has been observed
on day 14, indicating a higher rate of proliferation and growth on
G3-SPION-PCL compared with PCL scaffold. The better cell growth on
G3-SPION-PCL can be attributed to G3-SPIONs nanoparticles ,which endue
more hydrophilicity and positive surface charge to the prepared
scaffold, leading to the improved cell growth and proliferation. It is
noticeable that the porous structure of the scaffolds causes the
migration of some seeded cells from the porous nanofibrous scaffold to
the TCP plate, resulting in lower OD value of the scaffolds compared
with TCP.
The potential of PCL and G3-SPION-PCL nanofibrous scaffolds on cell
attachment and ADMSCs proliferation rate was also evaluated by DAPI
staining on days 1 and 14. As shown in Fig. 5b, the scaffold containing
G3-SPIONs showed enhanced cell attachment ,and growth compared to the
neat scaffold, PCL. The MTT assay indicated that the G3-SPION-PCL
scaffold was more compatible than PCL and according to the images of
DAPI staining the number of attached cells to G3-SPION-PCL was much more
than PCL. It can be concluded that the presence of G3-SPIONs decreases
the fiber diameter, increases the surface to volume ratio,and affect
significantly on the cell attachment. Overall, surface topography,
hydrophilicity, and positively charged PAMAM dendrimers ,which were
partially exposed on the scaffold surface play a vital role in the
interaction of cells to the scaffold
(Gloria et al., 2013).
An electromagnetic bioreactor was used to induced EMF and enhance the
osteogenesis of ADMSCs mediated by incorporated SPIONs. Many studies
demonstrated that EMF has a positive effect on the osteoblast and
adipocyte differentiation of mesenchymal stem cells
(Wang, Wu, Yang, & Song, 2016). Possible
effects of scaffold modification via incorporation of the dendrimerized
SPIONs, in the presence of biochemical (OM), and biophysical signaling
factors (PEMF) on osteogenesis potential of ADMSCs was evaluated by
monitoring the expression of the osteogenic marker on days 7 and 14
after cell seeding.
ALP activity is known as an early marker of osteogenic differentiation.
The research studies have indicated that the ALP enzyme plays an
essential role in the initiation of mineralization
(Miron & Zhang, 2012). As shown in Fig.
6a, the presence of SPIONs, OM, and PEMF resulted in a significant
difference in ALP activity between G3-SPION-PCL/OM/PEMF and the other
group. The presence of both biochemical and biophysical factors
increased the ALP activity ,which is in line with the previous studies
reported on the positive effect of electromagnetic waves on the
osteogenic differentiation (Xia et al.,
2018). Dankova et al. (Daňková et al.,
2015) demonstrated that the PCL-MNPs scaffolds increased the ALP
activity significantly in cells cultivated on the PCL-MNPs on day 7 (OD
around 0.2). Here, higher ALP activity could be due to dendrimers coated
MNPs, which significantly promote adhesion density and osteoblast
proliferation via 3D structure and considerable positive charge. The
calcium deposition value, as the late marker of osteogenic
differentiation, was evaluated quantitatively by calcium content assay,
Fig. 6b. As shown, there were no significant differences between the
amount of calcium deposition in TCP and PCL scaffold. While the calcium
content of G3-SPION-PCL/OM, G3-SPION-PCL/PEMF, and G3-SPION-PCL/OM/PEMF
is significantly higher than TCP and PCL. Also, G3-SPION-PCL/OM/PEMF
significantly showed higher calcium content than the other groups,
confirming the synergistic effect of OM and PEMF in the osteogenic
differentiation of ADMSCs. Since the G3-SPION-PCL/OM/PEMF group
demonstrated the best ALP activity ,as well as calcium deposition in the
osteogenic differentiation, SEM analysis of this sample was performed to
investigate the cell adhesion, morphological features, , and shape of
the cells on days 1 and 14. As shown in Fig. 6c, no calcium
precipitation was observed on the first day, but upon differentiation on
day 14 led to a higher level of calcium deposition. Dendritic
architecture introduced benefits over linear polymers with the aim of in
vitro bone cell growth and adhesion (Joshi
& Grinstaff, 2008). Also, it was observed that the morphology of hMSCs
showed a significant change from a fibroblastic shape into a polygonal
shape during osteodifferentiation (Erices,
Conget, Rojas, & Minguell, 2002). This study confirms that the
rational design of dendritic architectures can result in the fabrication
of new scaffolds with improved properties. The effect of PEMF
and OM on the expression level of osteogenic transcription factors was
evaluated on day 14. As shown in Fig. 7, the ADMSCs seeded on the
G3-SPION-PCL scaffold in the presence of OM and exposure to PEMF
expressed the highest level of Osteocalcin and Runx2. Osteocalcin, as
the most plentiful non-collagenous bone matrix protein, is mainly
expressed by osteoblasts and serves a specific marker for the late
stages of bone differentiation. Whenever Osteocalcin is overexpressed,
the mineral species maturation will be accelerated and modulates
osteogenic differentiation of MSCs (Tsao
et al., 2017). The superposition of OM and PEMF resulted in significant
expression of this gene by ADMSC cultured on the G3-SPION-PCL scaffold.
Collagen type I (Col 1) is an early marker of bone differentiation and
the most abundant protein in the bone matrix. The result (Fig. 7)
demonstrated overexpression of Col 1 by the cells cultured on the
G3-SPION-PCL scaffold under the only PEMF exposure. Interestingly, this
result showed that PEMF exposure accelerates early-stage osteogenic
differentiation. However, in the combination with OM and PEMF exposure,
Col 1 expression did not show such elevation. Recent researches have
proved that PEMFs improve osteogenic differentiation of MSCs by
stimulating the mRNAs expression of osteogenic related genes such as
RunX2, ALP, and BMP2 (Chen et al., 2019).
Taking together, it could be concluded that the presence of G3-SPIONs in
the PCL scaffold along with simultaneously PEMF exposure and OM
increases the differentiation rate of ADMSCs to osteogenic linage and
can be a suitable candidate in bone tissue engineering.
Conclusion
In this study, SPIONs were synthesized by the co-precipitation method
and modified by G3-PAMAM dendrimer using a divergent method. The
nanoparticles were incorporated into the PCL scaffold by electrospinning
to produce a novel magnetic scaffold. The influence of G3-SPIONs
incorporation on the properties of the magnetic electrospun nanofibers
were investigated. MTT assay and DAPI staining confirmed enhancement in
growth and proliferation of stem cells seeded on magnetic G3-SPION-PCL
compared to the pure PCL. After that, the osteogenic differentiation of
the magnetic electrospun scaffolds under biochemical (OM) and
biophysical (PEMF) stimulations were investigated ,and analyzed by
calcium content assay, ALP activity and real-time PCR. Results
interestingly confirmed the superposition effect of G3-SPIONs and PEMF
to increase osteogenic differentiation of ADMSCs.
In conclusion, this study suggests that G3-SPION incorporated PCL
nanofibers could be considered as a potential magnetic scaffold for bone
tissue regeneration applications.